Our long-term goal is to elucidate molecular mechanisms of aberrant gene control in cancer and develop innovative treatment strategies to improve the survival of children with refractory cancers such as AML. The objective of this project, which is the next logical step towards this goal as part of our collaborative U54 consortium, is to define and therapeutically target the oncogenic gene expression control activity of NUP98 fusion protein complex in AML. Our central hypothesis is that NUP98 fusion proteins generate an oncogenic chromatin remodeling protein complex that induces leukemogenic gene expression, conferring aberrant leukemia cell growth and survival, and that its molecular mechanisms and therapeutic targets can be defined using an innovative integration of functional genomics and proteomics. This hypothesis is based on two essential preliminary studies that the Armstrong and Kentsis labs have recently completed (3, 6), providing the impetus for this project: 1) Discovery of the essential functional interaction between NUP98 fusion proteins and the MLL1 and NSL chromatin remodeling complexes in AML; and 2) development of strategies to define the architecture and therapeutic disassembly of cellular protein complexes. However, the precise mechanisms linking NUP98 fusion protein complex assembly and leukemogenic chromatin functions are not defined, constituting an important gap in knowledge that is required for the development of a rational therapeutic strategy to treat fusion oncoprotein-activated AML. Aim 1 will elucidate the mechanisms of leukemogenic chromatin remodeling using inducible degradation of NUP98-fusion proteins. In Aim 2, we will define the domains and interfaces involved in aberrant assembly of NUP98-fusion chromatin remodeling complexes using in situ cross-linking mass spectrometry and CRISPR domain scanning. And lastly, we will determine the anti-leukemia efficacy of targeted epigenetic and protein interaction therapies using preclinical mouse models. Successful completion of this proposal is expected to yield molecular mechanisms and effective therapies of NUP98 fusion oncoprotein and gene control in AML, thus providing essential insights into a fundamental problem that remains poorly understood. This research will have broad significance because fusion oncoproteins and oncogenic gene expression contribute to the majority of human cancers. Finally, the complementary interactions with other projects in this U54 consortium and development of effective molecular therapies targeting NUP98 fusion proteins in AML would constitute a transformative advance in the clinical care of patients with this disease, whose cure rates remain wholly inadequate with current therapy.